Interactions between Shh pathway regulators and fetal alcohol exposure in mice

描述（由申请人提供）：前脑无裂畸形（HPE）是一种常见的前脑和面中部出生缺陷，有环境和遗传原因。环境因素之一是母亲在怀孕早期饮酒。此外，已在人类HPE中鉴定了Sonic hedgehog（Shh）信号通路中的突变。突变携带者的HPE表型是高度可变的，其影响范围从前脑和面中部的严重缺陷到没有临床表现。这种变异性的原因尚不清楚，但可能还需要的因素包括胎儿发育期间的额外遗传影响或环境暴露。基因与环境的相互作用很可能与HPE这样的复杂疾病有关;然而，对此几乎没有模型。Cdo和Boc是细胞表面蛋白，其结合Shh并调节发育胚胎的特定区域中的通路信号强度。Cdo突变小鼠表现出具有品系特异性严重程度的HPE：129/Sv背景上的突变体表现出非常轻微的HPE，频率较低，而C57 BL/6背景上的突变体表现出更严重的形式，频率较高。Boc突变小鼠没有明显的影响，但129/Sv Cdo;Boc双突变体显示严重的颅面HPE特征。我们报告说，129/Sv小鼠对乙醇诱导的HPE有抵抗力;然而，80%的129/Sv Cdo突变胚胎在子宫内暴露于乙醇显示出强烈的颅面HPE，并与Cdo;Boc双突变体有惊人的相似之处。此外，乙醇处理的Cdo突变胚胎的前脑中Shh表达强烈减少。因此，Cdo的丧失和胎儿乙醇暴露协同产生HPE;这一事实，加上乙醇处理的Cdo突变胚胎与Cdo;Boc双突变体非常相似，认为Cdo的丧失和乙醇暴露对Shh信号传导具有协同作用。四肢和手指缺陷也与人类胎儿酒精暴露有关，C57 BL/6胚胎在子宫内暴露于酒精导致四肢和手指后部畸形。Shh是四肢和手指的所有后部图案所必需的。在遗传致敏的背景下，Boc而不是Cdo的缺失导致严重的手指缺陷。假设与129/Sv Cdo突变小鼠和HPE相似，129/Sv Boc突变小鼠将对乙醇诱导的肢体/手指异常敏感。关于乙醇如何破坏已知的肢体/手指图案调节器的信息相对较少，对乙醇处理的C57 BL/6和129/Sv小鼠的发育遗传分析将揭示这一过程。该提案的目的是：1）分析129/Sv Cdo突变小鼠中乙醇诱导的HPE; 2）鉴定129/Sv Cdo突变小鼠前脑中乙醇诱导的Shh表达下调的机制;以及3）分析Shh通路在乙醇诱导的肢体/手指图案缺陷中的作用。这些研究涉及分析一种新开发的基因-环境相互作用模型，该模型在一种常见的出生缺陷中定义了发育突变和乙醇之间的相互作用。这些信息可能会对公共卫生产生重要影响，并最终有助于对突变携带者的咨询。 公共卫生相关性：母亲在怀孕早期饮酒可能导致出生缺陷，这可能受到人类遗传变异的影响。该应用程序的重点是一个小鼠模型，其中子宫内酒精暴露和特定突变的组合导致前脑和中面的破坏性和常见的出生缺陷，称为前脑无裂畸形。这个模型的研究，并将其扩展到其他乙醇相关的出生缺陷，提出了，目的是阐明遗传易感性如何可能影响胎儿酒精暴露的结果。

DESCRIPTION (provided by applicant): Holoprosencephaly (HPE) is a common birth defect of the forebrain and midface with both environmental and genetic causes. Among the environmental factors is maternal alcohol consumption during early pregnancy. In addition, mutations in the Sonic hedgehog (Shh) signaling pathway have been identified in human HPE. The HPE phenotype of mutation carriers is highly variable, with the spectrum of effects ranging in a continuum from severe defects in the forebrain and midface to no clinical manifestation. The reason for this variability is un- known, but factors that may also be required include additional genetic influences or environmental exposures, during fetal development. Gene-environment interactions are likely to be involved in a complex malady like HPE; however, there are few models for this. Cdo and Boc are cell surface proteins that bind Shh and regulate pathway signaling strength in specific regions of the developing embryo. Cdo mutant mice display HPE with strain-specific severity: mutants on the 129/Sv background show very mild HPE with low frequency, while mutants on the C57BL/6 background show more severe forms at high frequency. Boc mutant mice are without overt effect, but 129/Sv Cdo;Boc double-mutants display severe craniofacial HPE features. We report that 129/Sv mice are resistant to ethanol-induced HPE; however, 80% of 129/Sv Cdo mutant embryos exposed in utero to ethanol display strong craniofacial HPE and bear a striking resemblance to Cdo;Boc double mutants. Furthermore, there is a strong reduction of Shh expression in the forebrain of ethanol-treated Cdo mutant embryos. Loss of Cdo and fetal ethanol exposure therefore synergize to produce HPE; this fact, plus that ethanol-treated Cdo mutant embryos are so similar to Cdo;Boc double-mutants, argues that loss of Cdo and ethanol exposure have a synergistic effect on Shh signaling. Limb and digit defects are also associated with human fetal alcohol exposure, and in utero ethanol exposure of C57BL/6 embryos results in malformations of the posterior aspects of limbs and digits. Shh is required for all posterior patterning of limbs and digits. Loss of Boc, but not Cdo, results in severe digit defects on a genetically-sensitized background. It is hypothesized that, similar to 129/Sv Cdo mutant mice and HPE, 129/Sv Boc mutant mice will be sensitized to ethanol-induced limb/digit anomalies. There is relatively little information available on how ethanol disrupts the known regulators of limb/digit patterning, and developmental genetic analyses of ethanol-treated C57BL/6 and 129/Sv mice will shed light on this process. The aims of this proposal are: 1) to analyze ethanol-induced HPE in 129/Sv Cdo mutant mice; 2) to identify mechanisms of ethanol-induced down-regulation of Shh expression in the forebrains of 129/Sv Cdo mutant mice; and 3) to analyze the role of the Shh pathway in ethanol-induced defects in limb/digit patterning. These studies involve analysis of a newly developed model of gene-environment interaction between a defined developmental mutation and ethanol in a common birth defect. Such information may have important public health impact and could ultimately aid in the counseling of mutation carriers. PUBLIC HEALTH RELEVANCE: Maternal alcohol consumption during early pregnancy can result in birth defects, and this may be influenced by genetic variation in the human population. This application focuses on a mouse model in which a combination of in utero alcohol exposure and a specific mutation leads to a devastating and common birth defect of the forebrain and midface, called holoprosencephaly. Studies on this model, and its extension to additional ethanol-associated birth defects, are proposed, with the goal of shedding light on how genetic susceptibility may influence the outcome of fetal alcohol exposure.